Hi.
I’ve been unsuccessfully trying to isolate DNA from neurons extracted from adult mouse brains for further downstream analysis.
First I’ve tried sorting neuronal nuclei (Approx. 105 NeuN+ nuclei/sample) (protocol by Nott et al Nat Protoc 2021), followed by DNA isolation and qPCR analysis. However, my PTHrP levels were always undetectable.
Then I moved to commercial kits, and bought the adult neuron isolation kit from Miltenyi. As I read, one would expect approx. 105 isolated neurons per adult brain. I did the whole trinity from Miltenyi: 1. Adult Brain Dissociation Kit, 2. Myelin Removal Beads II, 3. Adult Neuron Isolation Kit. However, again after following the protocols, and immediately isolating the DNA (Nucleospin tissue from MN) I did qPCR (sybr green) and again I did not detect PTHrP in my samples!
I’ve preformed DNA isolation for high yield and concentration in a final 60ul elution volume. Following DNA isolation, the samples were stored at +4C, and the qPCR was done the next morning.
In my control samples from other tissue, I can detect PTHrP which means the qPCR protocol and primers are working fine.
Am I doing something wrong with the DNA isolation and therefore losing my DNA? Is there an alternative method to get clean neuron populations for DNA isolation?
I would be grateful for any help!
Best,
Dear Abdus,
Yes. I've used the nanodrop spectrophotometer to quantify the DNA concentration in each sample. But each time it is undetectable.
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning